1. Field of the Invention
The present invention relates to a method for isolation and purification of amylases, and adsorbents used for the same as well as devices for the isolation and purification. More particularly, the present invention relates to cross-linked polyglucans or cross-linked glucose homooligomers suited for adsorbing glucoamylases and .beta.-amylase with high selectivity and a method for isolation and purification of amylases using them as well as devices for the method.
2. Related Art Statement
Glucose, isomerized glucose and maltose are currently produced industrially by hydrolysis of starches using .alpha.-amylase, glucoamylase or .beta.-amylase.
As such, glucoamylase and .beta.-amylase are extremely useful for industrially producing useful low molecular sweeteners from starches.
In general, enzymes are prepared by liquid culture of microorganisms capable of producing the enzymes, namely, enzyme-producing bacteria. Enzymes as reagents for research have been used after partial purification or purification to a high degree, by complicated processes for isolation and purification, in combination with salting out, ion exchange chromatography, electrophoresis, etc.
On the other hand, preparations of enzyme for industrial use are obtained as concentrates of culture filtrates concentrated as they are, or dry powders, or concentrates obtained by isolation and purification of the filtrates or dry powders of the concentrates.
However, the concentrates of culture filtrates or crude dry products contain large quantities of components of unpleasant odor or colored components which are contained in the culture filtrates. Therefore, upon isolation and purification of the desired reaction product, it is necessary to finally remove these components. Further in many cases, culture filtrates also contain proteases. In crude enzyme solutions such as culture filtrates or the like, which contain proteases in addition to amylases, the amylases are often decomposed and inactivated by the action of proteases, during the course of purification or upon progress of the desired reaction. For this reason, it is necessary to remove these unfavorable impurities, in producing useful materials utilizing reactions of enzymes such as amylases.
Thus, a heavy burden is loaded on steps of isolation and purification for products in subsequent reactions.
On the other hand, salting out and liquid chromatography which are known to be conventional methods for purification have a limit that is partial concentration. In order to enhance accuracy of isolation, these operational conditions, for example, kind of precipitating agent, concentration, pH, kind of filler, kind of adsorbent and desorbent, or the like, must be changed, and complicated processes in combination with these conditions must be used.
Moreover, the operations involve addition of salts in large quantities as precipitating agents or eluting agents, which also necessitates desalting operations upon subjecting to a subsequent step. Further, with respect to BOD wastes showing a salt concentration as high as several 10% which generate during the course of such purification steps, it is not so simple to treat the wastes.
From the foregoing, known methods for purification comprising these complicated steps are limited mainly to purification of reagents or the like for research use.
If there is developed an adsorbent capable of selectively adsorbing glucoamylase and .beta.-amylase known to be particularly difficult to be purified, irrespective of difference in structure of enzyme molecule due to difference in origin of enzyme, both enzymes could be concentrated and isolated from culture solutions at one step in a high purity.
Paying attention to the adsorption method using amylose (linear polymer of glucose, molecular weight of 100,000 to 400,000, number of glucose polymerized: 5.times. 10.sup.2 to 2.times.10.sup.3) as an adsorbent that is known to be a method of purification of .alpha.-amylase, the present inventors have attempted to apply the adsorption method to glucoamylase and .alpha.-amylase. However, its adsorption capacity is as extremely low as 1/10.sup.3 or less, as compared to the case of .alpha.-amylase. It is thus understood that the known method using amylose as an adsorbent is not practical for purification of glucoamylase and .beta.-amylase.